1. Field of the Invention
This invention relates to proteins having asymmetric hydrolase activity for 4-substituted 1,4-dihydropyridine derivatives, genes encoding the proteins, and the use of the proteins.
2. Description of the Related Art
Of 4-substituted 1,4-dihydropyridine derivatives, those in which two different substituents are attached to the 3- and 5-positions of the dihydropyridine ring have an asymmetric carbon atom at the 4-position thereof and exist in the form of two optical isomers. As a result of investigations on the biological properties of these compounds, it has recently been reported that there is a difference in pharmacological activity, in vivo dynamic behavior, safety and other properties between the optical isomers of each compound [K. Tamazawa et al., J. Med. Chem., Vol. 29, 2504 (1986)]. Where such a compound having an asymmetric carbon atom is used as a drug, the conception of administering only one isomer favorable for use as a drug is being popularized so that no extra burden may be imposed on the living body. From this point of view, various processes for the preparation of optically active 3,5-disubstituted derivatives of 4-substituted 1,4-dihydropyridines are being investigated.
As a general method for the synthesis of optically active 4-substituted 1,4-dihydropyridine derivatives (in particular, 3,5-dicarboxylic acid monoesters), there is known a process comprising the steps of using a (4R)-1,4-dihydropyridine-3 or 5-carboxylic acid monoester as an intermediate and introducing a desired ester group thereinto [A. Ashimori et al., Chem. Pharm. Bull., Vol. 39, 108 (1991)]. Well-known methods for the preparation of such optically active intermediates [i.e., (4R)-1,4-dihydropyridine-3,5-carboxylic acid monoesters] include the chemical process of Shibanuma et. al. [Chem. Pharm. Bull., Vol. 28, 2809 (1980)], as well as the enzymatic process of Achiwa et al. [Tetrahedron Letters, Vol. 32, 5805 (1991)] and the enzymatic process of Charles J. Sih et al. [Tetrahedron Letters, Vol. 32, 3465 (1991)].
However, the above-described processes do not always make it possible to prepare the desired compounds efficiently. Consequently, the present inventors have proposed a simpler and efficient process for converting a 4-substituted 1,4-dihydropyridine-3,5-dicarboxylic acid diester to the corresponding 3,5-dicarboxylic acid monoester efficiently by using a culture of a microorganism such as bacteria of the genera Streptomyces, Paecilomyces, Botryodioplodia and Alternaria (see the pamphlet of PCT International Publication No. 94/05637).
However, there still exists a need for the provision of a more efficient means for the preparation of optically active 4-substituted 1,4-dihydropyridine-3,5-dicarboxylic acid monoesters.
Accordingly, an object of the present invention is to provide isolated genes which are conducive to the efficient preparation of the aforesaid optically active compounds, expression plasmids containing such genes, transformants obtained by using such plasmids, and proteins obtained by culturing such transformants and having asymmetric hydrolase activity for 4-substituted 1,4-dihydropyridine derivatives, as well as means associated with the use thereof.